MicroRNAs (miRNAs) are a class of small, endogenous and non-coding RNAs able to negatively regulate gene expression by targeting specific messenger RNAs (mRNAs) and inducing their degradation or translational repression (Ambros, 2004; Bartel, 2009). A recent study has defined mRNA degradation as the predominant mechanistic effect of miRNA:mRNA targets (Guo el al., 2010). Several recent studies have assessed the direct role of miRNAs in vascular inflammation and in the development of vascular pathologies (Kartha and Subramanian, 2010; Urbich et al., 2008).
MiR-145 was shown to be abundantly expressed in the vessel wall (Cheng et al., 2009). MiR-145 is transcribed as a long pri-miRNA encoding both miR-143 and miR-145 on human chromosome 5 (Lio et al., 2010) and on mouse chromosome 18, regulated by a conserved SRF-binding site (Xin et al., 2009). Localization of miR-145 to the vessel wall demonstrated high expression in the smooth muscle layer in comparison with adventitial fibroblasts and endothelial cells (Cheng et al., 2009). For this reason, miR-145 is considered a smooth muscle cell phenotypic marker and modulator, able to regulate smooth muscle cell (SMC) maturation and proliferation, and vascular neointimal lesion formation through its target gene KLF-5 and its downstream signaling molecule, myocardin (Cheng et al., 2009; Elia et al., 2009).
Agonists within the TGF-β superfamily have been shown to active miR-143/145 cluster via a Smad-dependent pathway (Davis-Dusenbery et al., 2011; Long et al., 2011). Moreover the analysis of miR-145, miR-143 and miR-143/145 knock-out (ko, −/−) mice showed a noticeably thinner smooth muscle layer of the aorta and other peripheral arteries, due to a reduced SMC size induced by a disruption of actin filaments (Elia et al., 2009). This leads to moderate systemic hypotension and the absence of neointima formation in response to injury in miR-145 −/− mice (Xin et al., 2009). Moreover, vascular smooth muscle cells (VSMCs) isolated from single and double ko animals showed hyperproliferative activity and a higher ability to migrate towards platelet-derived growth factor (PDGF), a known chemoattractant for VSMCs (Elia et al., 2009; Xin et al., 2009). Furthermore, a pharmacological analysis of the vasculature of miR-143(145) ko mice revealed a blunted response to vasopressive stimuli (Elia et al., 2009; Xin el al., 2009). Taking together, these findings show a dedifferentiated phenotype of VSMCs in miR-145 ko and miR-143/145 double ko mice.
Pre- and mature forms of miR-145 are also elevated in lung tissues and isolated pulmonary artery smooth muscle cells (PASMCs) obtained from pulmonary arterial hypertension (PAH) patients with a mutation in the BMPR2 gene compared to controls. One of the main histopathological features common to all forms of PAH is the accumulation of cells expressing smooth muscle specific α-actin (SMA) in peripheral pulmonary arteries. This includes the appearance of SMA-positive cells in the neointima and the extension of SMA-positive cells into precapillary pulmonary arterioles that are normally devoid of smooth muscle (Mandegar et al., 2004). The cellular processes responsible for the muscularization of this distal part of the PA are not clear, but these observations suggest a central role for PASMCs in the development of PAH.
Accordingly, miR-145, with a role in vascular remodeling, represents a therapeutic target the development of effective treatments for conditions associated with aberrant smooth muscle cell proliferation, such as in neointima formation, restensosis, hypertension, systemic hypertension and PAH. However, delivery of an antisense-based therapeutic targeting miR-145 can pose several challenges. The binding affinity and specificity to miR-145, efficiency of cellular uptake, and nuclease resistance are all factors in the delivery and activity of an oligonucleotide-based therapeutic. For example, when oligonucleotides are introduced into intact cells they are typically attacked and degraded by nucleases leading to a loss of activity. Thus, a useful antisense therapeutic should have good resistance to extra- and intracellular nucleases, as well as be able to penetrate the cell membrane.
Thus, there is a need for improved chemical modifications for stable and efficacious oligonucleotide-based inhibitors of miR-145. The present invention meets this need and provides related advantages as well.